Bacterial adhesion to teeth is the first step in the formation of dental plaque, a causative factor of dental diseases. Bacteria that bind must possess unique molecules that confer a selective advantage during colonization. This proposal aims to elucidate the role of such molecules which enable Streptococcus sanguis, an early colonizer of tooth surfaces, to adhere to salivary pellicle. S. sanguis 12 adheres to saliva-coated hydroxyapatite (SHA) surfaces through two distinct adhesin-receptor interactions. One interaction involves a bacterial surface lipoprotein (Streptococcus sanguis adhesin B: a 36-kDa protein designated SsaB) which mediates binding to an unidentified salivary molecule. The second interaction involves a sialic acid-binding lectin that interacts with a neuraminidase-sensitive receptor on SHA. Nucleotide and amino-acid sequence analyses of SsaB and SsaB-like proteins from Streptococcus parasanguis FW213 and Streptococcus gordonii PK488 have revealed that: (a) these proteins are lipoproteins with a high degree of homology (80- 90%); (b) SsaB-like proteins are prevalent in most species of viridans streptococci that are early colonizers; and (c) they may have evolved from a binding-lipoprotein-dependent transport system of Gram-positive bacteria. The goal of this proposal is to understand these adhesive interactions at the molecular level. Aim I. To identify and characterize salivary receptor(s) for S. sanguis 12. Saliva will be fractionated by gel filtration and ion-exchange chromatography and coated onto HA beads and assayed for adhesion-modulating activities. Fractions containing activity will be used in SsaB-affinity chromatography and SsaB-adhesion inhibition assays to identify and characterize the complementary salivary receptor present on SHA for SsaB. Aim II. Adhesin-negative mutants will be constructed by insertional inactivation of the gene(s) for the adhesin. These will be used for complementation studies utilizing streptococcaI shuttle vectors to reintroduce altered adhesin genes constructed in aim Ill. Aim III. The binding domain of SsaB will be identified by constructing nested deletion mutants and by linker insertion mutagenesis. Mutant SsaB proteins will be purified and analyzed for their ability to inhibit binding of S. sanguis 12 to SsaB salivary receptor coated-HA or SHA. The binding domain will be further defined by incorporating site-specific amino-acid changes during amplification using the polymerase chain reaction (PCR). Mutated SsaB proteins will be analyzed for inhibition of binding to SHA and by complementation studies to assess the effect of specific changes on the adhesin of S. sanguis 12 binding to SHA. These studies can be used to devise strategies to inhibit plaque formation.